The long term goal of this research project is to define in molecular terms the mechanisms controlling expression of a rat gene that is under multihormonal and developmental control and has recently found to be activated during the immediate-early growth response, namely gene 33(g33). The focus will be primarily on rat hepatoma and other tissue culture cell lines. The effect of agents such as: (1) insulin, a growth factor in certain tissues and established cell lines; (2) cAMP, the well known second messenger, which can exert positive control on cell growth in certain cell types, but a negative control in others; (3) phorbol esters; tumor promoters which activate protein kinase C(PKC), which may lead to cell proliferation in some cell types; (4) and serum, a known mitogenic agent; will be analyzed to identify and characterize the DNA sequences involved in the acceleration of transcription of g33. All of these agents, in addition to steroid hormones and plant lectins, have been shown to increase g33 levels in rat H4IIE hepatoma cells with the exception of cAMP, whose positive effect has been studied only in rat adult and fetal liver. The response of g33 to these agents in cultured hepatoma cell provides a good model for analysis of their respective mechanisms of signal transduction. The elucidation of the mechanism of action of growth factors has emerged as one of the fundamental problems in biology and may prove crucial for understanding the unrestrained proliferation of cancer cells. To identify and characterize the DNA sequences involved in g33 regulation by the above mentioned agents the fusion gene approach (employing the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter gene) will be utilized in stable transfection and in-vitro-transcription expression assays as well as DNA binding protein studies (genomic footprinting, gel retardation and DNase I footprinting). Different regions of g33's 5'-flanking DNA will be joined to the coding region of the CAT gene, and deletion as well as mutational analysis will be performed, with emphasis on changes in basal level of expression and/or response to extracellular agents. The role of protein phosphatases in the regulation of g33 expression will be studied using okadaic acid, a specific inhibitor of type 1 and 2a protein phosphatases.